Every year, hundreds of thousands of Americans sprain, tear, or rupture ligaments and tendons of the knee, elbow, hand, shoulder, wrist and jaw (Langer et al., Science 260: 920-926 (1993)). Of particular importance is the anterior cruciate ligament of the knee. More than 200,000 people in the U.S. alone, will tear or rupture their anterior cruciate ligament (ACL) each year (Albright et al., 1999. Chapter 42-Knee and Leg:Soft-Tissue Trauma. In Orthopaedic Knowledge Update 6. American Academy of Orthopaedic Surgeons).). The ACL serves as a primary stabilizer of anterior tibial translation and as a secondary stabilizer of valgus-varus knee angulation, and is often susceptible to rupture or tear resulting from a flexion-rotation-valgus force associated with sports injuries and traffic accidents. Ruptures or tears often result in severe limitations in mobility, pain and discomfort, and the loss of an ability to participate in sports and exercise. Failures of the ACL are classified in three categories: (1) ligamentous (ligament fibers pull apart due to tensile stress), (2) failure at the bone-ligament interface without bone fracture, and (3) failure at the bone-ligament interface with bone fracture at the attachment site of bone and ligament. The most common type of ACL failure is the first category, ligamentous.
It is widely known throughout the medical community that the ACL has poor healing capabilities. Total surgical replacement and reconstruction are required when injury to the ACL involves significant tear or rupture. Four options have been utilized for repair or replacement of a damaged ACL: (1) autografts, (2) allografts, (3) xenografts, and (4) synthetic prostheses (degradable and non-degradable). To date, no surgical repair procedure has been shown to restore knee function completely, and novel treatment options would likely benefit a large number of patients.
The problems associated with the use of synthetic ACL replacements, along with the limited availability of the donor tissue, have motivated research towards the development of functional and biocompatible equivalents of native tissues. This shift from synthetic to biologically-based ACL replacements first applied in early studies in which collagenous ACL prostheses were prepared as composite structures consisting of reconstituted type I collagen fibers in a collagen I matrix with polymethylmethacrylate bone fixation plugs, and used as anterior cruciate ligament replacement tissues in rabbits (Dunn et al., Am. J. Sports Medicine 20: 507-515 (1992)). Subsequent studies incorporated active biological components into the process, such as ligament fibroblasts seeded on cross-linked collagen fiber scaffolds that were used as ligament analogs (Dunn et al., J. Biomedical Materials Res. 29: 1363-1371 (1995); Dunn, M. G., Materials Res. Soc. Bulletin, Nov: 43-46 (1996)), and suggested that structures approximating native ligaments can be generated.
A tendon gap model, based on pre-stressed collagen sutures seeded with mesenchymal stem cells provided improved repair of large tendon defects (Young et al., 1998). Goulet et al. modified the collagen-fibroblast system by using ligament fibroblasts in non-cross-linked collagen, with bone anchors to pre-stress the tissue and facilitate surgical implantation (Goulet et al., Tendons and Ligaments. In Principles of Tissue Engineering, Ed. R. Lanza, R. Langer, W. Chick. R. G. Landes Co. pp 633-643, R. G. Lanz Co. and Academic Press, Inc., San Diego, Calif. (1997)). Passive tension produced by growing the new ligament in a vertical position induced fibroblast elongation and the alignment of the cells and surrounding extracellular matrix.
Silk has been shown to offer new options for the design of biomaterials and tissue-engineering scaffolds with a wide range of mechanical properties (Sofia, S., et al., J. Biol. Mat. Res. 54: 139-148 (2001). For example, the dragline silk from the orb weaving spider, Nephilia clavipes, has the highest strength of any natural fiber, and rivals the mechanical properties of synthetic high performance fibers. Silks also resist failure in compression, are stable at high physiological temperatures, and are insoluble in aqueous and organic solvents. In recent years, silks have been studied as a model for the study of structure-function relationships of fibrous proteins. The manipulation of silk genes, both native and artificial versions, has provided insight into silk protein expression, assembly, and properties. Thus, biocompatibility, the ability to engineer the materials with specific and impressive mechanical properties, and a diverse range of surface chemistries for modification or decoration suggests that silk may provide an important class of biomaterial. Recent studies by the inventors of the present invention have demonstrated the successful attachment and growth of fibroblasts on silk films from silkworm silk of Bombyx mori. 
Tissue engineering can potentially provide improved clinical options in orthopaedic medicine through the in vitro generation of biologically based functional tissues for transplantation at the time of injury or disease. Further, adult stem cells are becoming increasingly recognized for their potential to generate different cell types and thereby function effectively in vitro or in vivo in tissue repair. (Sussman, M. Nature 410: 640 (2001). The knee joint geometry and kinematics and the resultant effects on ACL structure must be incorporated into the construct design if a tissue engineered ACL generated in vivo is to successfully stabilize the knee and function in vivo. A mismatch in the ACL structure-function relationship would result in graft failure.
To date, no human clinical trials have been reported with tissue culture bioengineered anterior cruciate ligaments. This is due to the fact that each approach has failed to address one or more of the following issues: (1) the lack of a readily available cell or tissue source, (2) the unique structure (e.g., crimp pattern, peripheral helical pattern and isometric fiber organization) of an ACL, and (3) the necessary remodeling time in vivo for progenitor cells to differentiate and/or autologous cells to infiltrate the graft, thus extending the time a patient must incur a destabilized knee and rehabilitation. The development of a matrix for generating more fully functional bioengineered anterior cruciate ligaments would greatly benefit the specific field of knee reconstructive surgery, and would also provide wider benefits to the overall field of in vitro tissue generation and replacement surgery.